Methods of identifying therapeutic compounds in a genetically defined setting

ABSTRACT

The invention provides a method of identifying therapeutic compounds in a genetically defined setting. The method consists of contacting a cell indicative of a pathological condition from a diseased individual and a cell from a genetically related normal individual with a plurality of candidate therapeutic compounds under suitable assay conditions, and identifying a compound that preferentially alters a predetermined property of the cell from the diseased individual.

This application is a continuation of U.S. Ser. No. 10/357,556, filedFeb. 3, 2003, which is a continuation of U.S. Ser. No. 09/568,595, filedMay 10, 2000, which claims the benefit of priority of U.S. ProvisionalApplication No. 60/304,193, filed May 10, 1999, which was converted fromU.S. Ser. No. 09/309,468, each of which the entire contents isincorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to the fields of medicine andpharmacology and, more specifically, to methods of identifyingtherapeutic compounds in a genetically defined setting.

In the past, the process of discovering novel therapeutic compounds wasslow and laborious, and usually involved administering individuallysynthesized compounds to experimental animals in the hope of observing atherapeutic effect. Recently, significant advances have been made inmedicinal chemistry, resulting in the development of combinatorialchemistry methods that allow the rapid production of enormous librariesof structurally distinct compounds. Additionally, due to recent progressin understanding the underlying molecular mechanisms of many diseases,it has become possible to develop in vitro assays to rapidly screencandidate therapeutic compounds. Automation of these assays usingcomputer-controlled robotic systems in high throughput screening methodshas made it possible for biotechnology companies to screen millions ofcompounds per year.

The identification of therapeutic compounds using automated screeningmethods requires the development of in vitro assays that accuratelypredict the therapeutic potential of a compound identified by the assayfor treatment of the particular pathological condition. So far, currentdrug screening methods have fallen short of this goal. For example, avariety of cell-free assays have been developed that focus oninteractions of candidate compounds with isolated target molecules. Suchassays have been shown to be of limited value, since neither the bindingproperties nor the expected biological properties of the compounds haveusually proven to be relevant in vivo.

In an attempt to overcome the limitations of cell-free assays, a varietyof cell-based assays have recently been developed. Such assays detectparticular cellular functions believed to be relevant to the underlyingdisease mechanism. To date, however, most cell-based assays forscreening candidate therapeutic compounds have used established celllines. Established cell lines, as evidenced by their ability to bepropagated indefinitely in culture, are highly abnormal and are oftenneoplastically transformed. Therefore, screening assays using suchabnormal cell lines are poorly predictive of the therapeutic efficacy ofcompounds for affecting cell function in an individual.

Additionally, current cell-based assays to identify therapeuticcompounds generally use cell lines established from a single individual,or cell lines established from unrelated normal and diseasedindividuals. Screening assays using cells from unrelated individuals arelikely to identify compounds that alter a cellular function related tothe genetic differences between the individuals, rather than compoundsthat alter a cellular function relevant to the underlying diseasemechanism.

Therefore, there exists a need for improved methods of screeningcandidate therapeutic compounds. Ideally, such methods would userelevant cells and assay conditions so as to be highly predictive of thetherapeutic efficacy of the compounds. The present invention satisfiesthis need and provides related advantages as well.

SUMMARY OF INVENTION

The invention provides a method of identifying a therapeutic compoundpotentially effective against a predetermined pathological condition.The method consists of contacting a cell indicative of the pathologicalcondition from a diseased individual with a plurality of candidatetherapeutic compounds under suitable assay conditions, and alsocontacting a cell indicative of the pathological condition from a normalindividual, who is genetically related to the diseased individual, withthe plurality of compounds under the same assay conditions. A compoundfrom the plurality of compounds that preferentially alters apredetermined property of the cell from the diseased individual isidentified, and characterized as a therapeutic compound potentiallyeffective against the pathological condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a signal transduction pathway in a normal individual (PanelA) and two diseased individuals (Panels B and C).

FIG. 2 shows two signal transduction pathways that together produce asignal in an assay of a predetermined property.

FIG. 3 shows a method of mapping disease-associated gene products toloci of a signal transduction pathway.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to a method of identifying therapeuticcompounds effective against a variety of pathological conditions. Themethod is advantageous in using primary cells obtained from geneticallyrelated normal and diseased individuals to screen candidate compounds.Therefore, variables due to genetic heterogeneity and abnormalities ofestablished cell lines are minimized, and compounds identified by themethod will have a high likelihood of being therapeutically effective inpatients.

Specifically, the invention provides a method of identifying atherapeutic compound potentially effective against a predeterminedpathological condition. The method consists of contacting a cellindicative of the pathological condition from a diseased individual witha plurality of candidate therapeutic compounds under suitable assayconditions, and also contacting a cell indicative of the pathologicalcondition from a normal individual, who is genetically related to thediseased individual, with the plurality of compounds under the sameassay conditions. A compound from the plurality of compounds thatpreferentially alters a predetermined property of the cell from thediseased individual is identified, and characterized as a therapeuticcompound potentially effective against the pathological condition.

As used herein, the term “plurality of candidate therapeutic compounds”is intended to mean 2 or more different candidate therapeutic compounds.Such compounds can be used in a screening assay to identify one or morecompounds that affect a predetermined property of a cell from a diseasedindividual to a greater extent than a cell from a normal individual. Thenumber of different compounds in the plurality of compounds can bedetermined by those skilled in the art depending on the application ofthe method. For example, a smaller number of candidate compounds can beadvantageous if the type of compound that is likely to affect apredetermined property of the cell is known or can be predicted.Additionally, if the method is used to compare the efficacy of compoundsagainst cells from multiple normal or diseased individuals, it can bedesirable to practice the method using a smaller number of compounds.Therefore, a plurality of compounds can include 2, 3, 4, 5, 10 or more,20 or more, or 50 or more candidate compounds. Those skilled in the artalso understand that the method can be practiced with a single compound,if desired.

However, when the type of compound that is likely to affect apredetermined property of the cell is unknown, it is generallyunderstood that the larger the number of candidate therapeuticcompounds, the greater the likelihood of identifying a therapeuticcompound. Additionally, when the method is practiced using cells fromonly one or several diseased individuals, and one or several normalindividuals, it may be desirable to screen a large number of differentcompounds. Therefore, a plurality of candidate therapeutic compounds cancontain, for example, greater than about 10³ different compounds,preferably greater than about 10⁵ different compounds, more preferably,greater than about 10⁷ different compounds.

A candidate therapeutic compound can be a naturally occurringmacromolecule, such as a polypeptide, nucleic acid, carbohydrate, lipid,or any combination thereof. A candidate therapeutic compound also can bea partially or completely synthetic derivative, analog or mimetic ofsuch a macromolecule or, a small, synthetic molecule, such as an organicmolecule prepared by combinatorial chemistry methods. A candidatecompound can be detectably labeled or attached to a solid support, ifdesired, in a particular assay.

Methods for producing pluralities of compounds, including chemical orbiological molecules such as simple or complex organic molecules,metal-containing compounds, carbohydrates, peptides, proteins,peptidomimetics, glycoproteins, lipoproteins, nucleic acids, antibodies,and the like, are well known in the art and are described, for example,in Huse et al., U.S. Pat. No. 5,264,563; Francis et al., Curr. Opin.Chem. Biol. 2:422-428 (1998); Tietze et al., Curr. Biol., 2:363-371(1998); Sofia, Mol. Divers. 3:75-94 (1998); Eichler et al., Med. Res.Rev. 15:481-496 (1995); and the like. Libraries containing pluralitiesof candidate therapeutic compounds also can be obtained from commercialsources.

A therapeutic compound identified by a method of the invention ispotentially effective in preventing or treating a predeterminedpathological condition. As used herein, the term “pathologicalcondition” is intended to mean a disease state characterized by aberrantphysiological function or organization of cells, tissues or organs. Apathological condition can result, for example, from genetic ordevelopmental abnormalities, nutritional or environmental factors,infection, neoplasia, aging, altered immune or endocrine function,tissue damage, or any combination of these factors. The invention isparticularly amenable to identifying therapeutic compounds potentiallyeffective against pathological conditions with a known hereditarycomponent, and which affect a significant proportion of the population,such as, for example, asthma, cardiovascular disease, many types ofcancer, schizophrenia, dementia, obesity, and diabetes. The inventioncan also be practiced with respect to rarer or monogenetic diseases suchas, for example, diseases described in the Online Mendelian Inheritancein Man database (Center for Medical Genetics, Johns Hopkins University(Baltimore, Md.) and National Center for Biotechnology Information,National Library of Medicine (Bethesda, Md.) (1998)).

The methods of the invention are applicable to pathological conditionsthat affect all systems of the body, including, for example, thecardiovascular system, immune and hematopoietic system, respiratorysystem, hepatobiliary system, gastrointestinal system, endocrine system,urinary system, genital system, nervous system and musculoskeletalsystem. Certain pathologies are considered multisystem diseases andinclude, for example, systemic lupus erythematosus, systemic sclerosis,diabetes mellitus, and other inflammatory and metabolic disorders. Otherdiseases primarily or initially affect a single tissue or organ, such asa benign or malignant tumor of the breast, prostate, colon, lung, brainor ovary.

As used herein, the term “diseased individual” is intended to mean anindividual exhibiting, or is considered to be at elevated risk, comparedto the general population, of exhibiting signs or symptoms of apathological condition. In contrast, as used herein, the term “normalindividual” is intended to mean an individual who does not exhibit, oris considered to be at low risk of exhibiting, signs or symptoms of apathological condition. The signs, symptoms and genetic andenvironmental risk factors associated with different pathologicalconditions are known in the art and are described, for example, inStevens et al., ed., Pathology, Mosby: London (1995).

As used herein, the term “genetically related individual” is intended tomean an individual with a common ancestor within several generations. Amore distantly genetically related individual can share an ancestorwithin 10 or fewer generations, such as 8 or fewer generations,preferably 6 or fewer generations. A more closely genetically relatedindividual can share an ancestor within 4 or fewer generations, morepreferably 3 or fewer generations, most preferably 2 or fewergenerations. Genetically related individuals can be of the samegeneration, such as siblings, first cousins or distant cousins, or ofdifferent generations, such as a grandparents and grandchildren, parentsand children, and aunt or uncle and niece or nephew.

The method is advantageous in that the effect of a compound on a cellfrom a diseased individual and a cell from a normal individual,genetically related to the diseased individual, is compared. Therefore,genetic variables unrelated to the pathological condition are minimized,which could otherwise complicate the interpretation of the screeningassay. The more closely related a diseased and a normal individual are,the more likely it is that any difference observed is indicative of aneffect on the pathological condition, rather than an effect of thecompound on an irrelevant parameter. Therefore, when a method of theinvention is practiced using a cell from a single diseased and a singlenormal individual, it is preferable to obtain samples from closelyrelated individuals, such as siblings, more preferably fraternal twins,most preferably identical twins.

The invention can also advantageously be practiced using cells frommultiple individuals selected from a large family or geneticallyhomogeneous population that includes both normal and diseasedindividuals of varying degrees of relatedness. Using methods known inthe art, the degree of relatedness of the individuals within thepedigree, and the relative risk for each individual within the pedigreeof exhibiting the disease, can be established. The number of normal anddiseased individuals and their degree of genetic relatedness can bedetermined by those skilled in the art for a particular application ofthe method. For example, the method can be practiced using cells from 2,3, 4, 5, 10 or more, 20 or more, 50 or more, or 100 or more normalindividuals, depending on the number of compounds being screened, theassay format, the ease of obtaining the samples, and the statisticalsignificance required. Similarly, the method can be practiced usingcells from 2, 3, 4, 5, 10 or more, 20 or more, 50 or more, or 100 ormore diseased individuals.

In one embodiment, the invention can be practiced by obtaining cellsfrom diseased individuals who each exhibit the same degree of severityof the pathological condition. In another embodiment, the diseasedindividuals can exhibit a range of severities of the pathologicalcondition ranging, for example, from mildly affected to moderatelyaffected to severely affected. For example, with regard to a diseasesuch as cancer, cells could be obtained from diseased individuals whoexhibit differing disease severities ranging from, for example, benignhyperplasia or dysplasia, to neoplasia, to a highly metastatic tumor.

In a further embodiment, the diseased individuals can exhibit a range ofrisk of developing the pathological condition ranging, for example, froma moderate risk of developing the pathological condition, to a high riskof developing the pathological condition, to actually exhibitingsymptoms of the pathological condition. For example, in diseases with ahereditary component, the degree of risk can be related to the degree ofrelatedness to an affected individual. Furthermore, in diseases in whicha susceptibility locus or gene has been identified, the degree of riskcan be associated with the presence of none, one or two alleles of thesusceptibility locus or gene. Similarly, in diseases with anenvironmental component, such as exposure to cigarette smoke, toxins,radioactivity, nutritional factors and the like, the degree of risk canbe associated with the amount or extent of exposure to the environmentalcomponent.

Those skilled in the art can readily assess the degree of severity ofthe pathological condition for each individual and the degree of risk ofdeveloping the pathological condition for each individual, usingknowledge of the risk factors, pathological mechanisms, and clinicalsigns and symptoms of a given disease. Those skilled in the art can alsodetermine for a given application of the method the appropriate range ofdisease severity or risk, and the appropriate number of individuals witheach degree of disease severity or risk. Factors involved in determiningthe number of individuals include, for example, the statisticalsignificance of the data required, the qualitative or quantitativenature of the data obtained, and the availability of cells from a rangeof individuals for a given disease.

A method of the invention can advantageously be practiced using cellsfrom individuals from genetically homogeneous populations, such asgeographically isolated populations with relatively few founderindividuals, or populations that are isolated for cultural or religiousreasons. Isolated populations have had relatively little inwardmigration or intermarriage, and a result, most of the population isdescended from the original founder individuals. Therefore, there is anincreased likelihood that differences in the ability of therapeuticcompounds to alter a property of cells of diseased and normalindividuals will be related to the disease mechanism, rather than tocell-to-cell variations resulting from different genetic backgrounds.Additionally, environmental variation is likely to be minimal withinisolated populations. Genetically homogeneous populations alsoadvantageously include individuals with varying degrees of geneticrelatedness, from distantly related to closely related. Therefore, therelative effect of a therapeutic compound on a property of a cell can bestatistically analyzed and correlated with the degree of geneticrelatedness.

Examples of genetically homogeneous populations of individuals are knownin the art and include, for example, geographically isolatedpopulations, such as island populations. Preferably, geneticallyhomogeneous populations of individuals have extensive and accuratemedical records and detailed genealogical records. Geneticallyhomogeneous populations with extensive medical and genealogical recordsare well known in the art and include, for example, the population ofIceland, populations of the Scandinavian countries, the Mormonpopulation of Utah, and the Amish and Hutterite populations of NorthAmerica.

For certain diseases, epidemiological studies have been conducted amonggenetically homogeneous populations with an increased incidence of thedisease. Therefore, a method of the invention can be practiced usingcells obtained from diseased and normal individuals within suchpopulations to identify therapeutically effective compounds against suchdiseases. As several non-limiting examples, it is known in the art thatthe population of Tristan de Cunha has an increased prevalence ofasthma, as described in Zamel et al., Am. J. Respir. Crit. Care Med.153:1902-1906 (1996); that the Pima Indians have an increased frequencyof non-insulin dependent diabetes mellitus, as described in Bogardus etal., J. Cell Biochem. 48:337-343 (1992); that the population of FinnishNorth Karelia has an increased incidence of hypercholesteremia andcoronary heart disease, as described in Vuorio et al., Arterioscler.Thromb. Vasc. Biol. 17:3127-3138 (1997); and that the population of theCentral Valley of Costa Rica has increased prevalence of bipolardisorder, as described in Sheffield et al., Trends in Genetics14:391-396 (1998). Other genetically homogeneous populations susceptibleto particular pathological conditions of interest are known or can bedetermined by those skilled in the art.

The method is practiced by contacting a cell indicative of thepredetermined pathological condition with the plurality of candidatetherapeutic compounds under suitable assay conditions. As used herein,the term “cell indicative of a pathological condition” is intended tomean a cell that has, or can be made to have, one or more properties ina diseased individual that is detectably altered relative to a cell ofthe same histological origin from a normal individual. The term “a cell”is intended to include single cells, as well as pluralities of cells ofthe same or different histological type present in a cell suspension,cell culture, or tissue sample. The type and number of cells to use toidentify a therapeutic compound will depend on the particularpathological condition and the assay used, and can be determined bythose skilled in the art for a given application of the method.

A cell indicative of a pathological condition can be selected from atissue or organ affected, or most affected, in the particular disease.Alternatively, a cell indicative of a pathological condition can beselected from an apparently unaffected tissue of a diseased individual.Many diseases, such as a genetic or multisystemic disorders, will bemanifested in a variety of different tissues and cell types.Accordingly, it is not always necessary to know or to determine anaffected, or the most affected, cell or tissue. Therefore, a cellindicative of many pathological conditions can be obtained from anyconvenient source. A factor to be determined in obtaining cells,particularly from a large number of normal and diseased individuals, isthe ability to obtain the cells using minimally invasive methods.Therefore, cells from both normal and diseased individuals can readilybe obtained, for example, from fluids such as the blood, lymph, urine orbreast milk, or from accessible tissues such as the skin, hairfollicles, cervix or cheek. Additionally, cells can readily be obtainedfrom both normal and diseased individuals using slightly more invasiveprocedures, such as punch biopsies of the breast or muscle, or from thebone marrow or cerebrospinal fluid. Depending on the need and theavailability of an appropriate surgical procedure, cells fromessentially any organ or tissue of the body can be obtained fromgenetically related individuals and used in the methods of theinvention.

Those skilled in the art can readily determine which cells areindicative of a pathological condition, which cells are appropriatecontrol cells from normal individuals, and what is the most desirablesource of such cells. Additionally, methods of obtaining, storing,culturing, and manipulating cells to ensure the introduction of theminimal amount of irrelevant variations between samples are well knownin the art.

A cell indicative of a pathological condition can be a primary cell ortissue sample obtained directly from an individual. If desired,depending on the assay conditions employed, a cell indicative of apathological condition can also be modified or altered from how it wasinitially obtained from the individual. For example, a cell indicativeof a pathological condition can be a primary cell disaggregated fromconnective tissue and irrelevant cells using known methods, such as, forexample, enzymatic digestion and biochemical separation. Likewise, acell indicative of a pathological condition can be a cell separated fromother cells using affinity separation methods known in the art. As anexample, flow cytommetry or antibody panning methods can be used toselect a population of cells expressing a detectable surface marker suchas, for example, CD4, CD8, CD34 or CD38.

Additionally, a cell indicative of a pathological condition can be acell propagated in culture, using methods known in the art, for severalgenerations. Depending on the assay conditions employed, and asdescribed below, such a cell can also be a cell that has been transducedor transfected with a nucleic acid encoding an expressible reporterconstruct, or contacted with a detectable molecule such as aradiolabeled compound or fluorochrome.

A cell from one or more diseased individuals and a cell from one or morenormal individuals, selected as described above, are each contacted,either sequentially or simultaneously, with a candidate therapeuticcompound under suitable assay conditions. As used herein, the term“suitable assay conditions” is intended to mean conditions under which aparticular assay, such as an assay described below, will identify acompound that alters a predetermined property of a cell. Suitable assayconditions take into account factors such as the concentration of thecompound, the duration of contact with the compound, the temperature andbuffer conditions, the method of contact, whether or not cell viabilityis required, and the detection format. Suitable assay conditions dependon the cell type, the predetermined property to be detected, thepathological condition, and the number of compounds being screened.Assay conditions to identify compounds that alter predeterminedproperties of cells are known in the art or can be readily determinedfor a particular application of the method.

The method involves contacting a cell from a diseased individual and acell from a genetically related normal individual under suitable assayconditions with a candidate compound, such that the effect of thecompound on the predetermined property can be determined and a compoundthat preferentially alters the predetermined property of the cell fromthe diseased individual can be identified. The term “preferentiallyalter,” as used herein, is intended to mean qualitatively orquantitatively changing the predetermined property of a cell from adiseased individual, relative to the same property of a cell from anormal individual.

As used herein, the term “predetermined property” is intended to mean aproperty that is known to be, or is considered by those skilled in theart to be, a credible indication of the particular pathologicalcondition. The predetermined property to be detected can be chosen bythose skilled in the art using knowledge of the underlying pathologicalmechanisms that are associated with the pathological condition. Apredetermined property consistent with a method of the invention can bea biological process, a functional activity, or a structural property ofthe cell, so long as it is considered to be associated with thepathological condition and can be qualitatively or quantitativelydetected in a cell-based in vitro assay.

For example, a predetermined property associated with a pathologicalcondition can be a biological process such as cell proliferation,adhesion, differentiation, motility or apoptosis. Therefore, anappropriate assay would detect the ability of a compound topreferentially increase or inhibit such a process in cells from adiseased individual. Such biological assays generally involve initiallyviable cells, and assay conditions consistent with cell viability wouldbe chosen.

An as example, a therapeutic agent potentially effective against cancercould be identified by screening candidate compounds against cells froman individual having, or a risk of developing, a neoplastic tumor, andagainst normal cells from related normal individuals. Compounds thatpreferentially exhibit cytotoxic or cytostatic activity against cellsfrom the diseased individual, as determined by a reduction in cellnumber or viability, would be characterized as therapeutic compoundsthat are potentially effective against cancer.

A predetermined property associated with a pathological condition canalso be a functional activity, such as, for example, altered productionor turnover of a second messenger, GTP hydrolysis, influx or efflux ofions or amino acids, altered membrane voltage, increased or decreasedprotein phosphorylation, altered activity of an enzyme, alteredprotein-protein interactions, relocalization of a protein within thecell, or induction of gene expression, in response to contacting thecell with a therapeutically effective compound. Assays to detectalterations in these functional activities are well known in the art orcan be readily adapted to a novel predetermined property relevant to adisease of interest. Such assays are described, for example, in Gonzalezet al., Curr. Opin. in Biotech. 9:624-631 (1998) and in Jayawickreme etal., Curr. Opin. Biotech. 8:629-634 (1997), and in references reviewedtherein.

Often assays to detect a relevant functional activity involve firstcontacting the cell with a detectable biosensor, such as a fluorescentcalcium indicator, green fluorescent protein, a fluorophore, aradiolabeled compound, or a chemiluminescent indicator, either alone orlinked to another molecule such as an amino acid, peptide,oligosaccharide, nucleotide or nucleic acid. Additionally, such assayscan involve first transducing the cells with a promoter-reporter nucleicacid construct such that, for example, β-lactamase, luciferase, greenfluorescent protein or β-galactosidase will be expressed in response tocontacting the cell with a therapeutically effective compound.Appropriate assays to detect functional activities preferentiallyaltered in a diseased cell by a therapeutic compound can be determinedby those skilled in the art based on knowledge of the underlyingpathological mechanisms, such as knowledge of the signal transductionpathway or molecular interactions that underlie the pathology.

A predetermined property associated with a pathological condition canalso be a structural property, such as the altered ability of a compoundto bind a cell from a diseased individual. Therefore, a method of theinvention can detect a compound that interacts with receptors present inincreased or decreased abundance on the surface of cells from diseasedindividuals, or a compound that interacts with increased or decreasedaffinity with receptors present in equal abundance on the surface ofcells from normal and diseased individuals. Such compounds can be eitheragonists or antagonists of the receptor.

Assays suitable for detecting various binding interactions are known inthe art and include, for example, fluorescence correlation spectroscopy(FCS) and scintillation proximity assays (SPA), which are reviewed, forexample, in Major, J. Receptor and Signal Transduction Res. 15:595-607(1995); and in Sterrer et al., J. Receptor and Signal Transduction Res.17:511-520 (1997). Other assays for detecting binding interactionsinclude, for example, ELISA assays, FACS analysis, and affinityseparation methods which are described, for example, in Harlow and Lane,Eds., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory(1988). Such assays can often be performed with either viable ornon-viable cells.

If desired, a method of the invention can be practiced using an assaywherein one or several steps, such as cell manipulation, culture platemanipulation, contacting the cells with the compounds, detection of thepredetermined property, or statistical analysis of the data, areautomated. Such automation advantageously provides for high throughputscreening of candidate therapeutic compounds, often using smallernumbers of cells and smaller amounts of compounds and reagents thanmanual assays. Those skilled in the art can determine for a particularapplication of the method whether it would be advantageous to automateone or more steps of the screening assays. Methods of automating theassays described herein are well known in the art.

When the invention is practiced with a large number of compounds or withcells from a large number of individuals, or both, such as in ahigh-throughput screening format, the efficacy of compounds in alteringa predetermined property in cells from normal and diseased individualscan be rank ordered and analyzed using known statistical methods. Forexample, when the invention is practiced with regard to a panel of cellsfrom individuals with a range of disease severities, or with a range ofdisease risk, the effect of a compound in altering a predeterminedproperty can be correlated with the disease severity or risk of thecells it affects, using, for example, statistical methods known withinthe art. Therefore, the method provides a means of rapidly identifyingcompounds that are potentially effective in preventing or treating aparticular pathological condition in all individuals, most individuals,or only those individuals with a given degree of disease severity orrisk. Additionally, the method can provide a means of comparing andranking the potential of multiple compounds to be effective against aparticular pathological condition in multiple individuals with varyingdegrees of disease severity or risk.

Statistical methods for analyzing the data obtained using the methods ofthe invention can be similar to methods used to map disease associatedgenes. Such statistical methods are described, for example, in Weeks etal., Trends in Genetics 11:513-519 (1995), in Taylor et al., MethodsMol. Biol. 68:11-25 (1997), and in Lynch and Walsh, Genetics andAnalysis of Quantitative Traits, Sinauer Associates, Inc., Sunderland,Mass. (1998).

The method of identifying a therapeutic compound that preferentiallyalters the predetermined property of a cell from a diseased individual,from the plurality of candidate compounds, will depend on the number ofcompounds being tested and the particular assay employed. For example,the method of the invention can be repeated by subdividing pools ofcompounds into smaller pools, until a single compound that reproduciblypreferentially alters a predetermined property of a cell from a diseasedindividual is identified. Alternatively, a compound that preferentiallyalters a predetermined property of the cell can be isolated away fromthe cell it affects and its identity determined. Additionally, acompound that preferentially alters a predetermined property of the cellcan be identified by virtue of an inherent characteristic structural orfunctional property, or by virtue of a distinguishing label. These andother methods of identifying a potentially effective therapeuticcompound resulting from practice of the method of the invention areknown in the art.

A therapeutic compound identified by a method of the invention ispotentially effective against the predetermined pathological condition.As used herein, the term “potentially effective” is intended to meanthat a compound identified by a method of the invention has an increasedlikelihood, relative to a randomly chosen compound, to be effective inpreventing or treating the pathological condition in vivo. Determiningthe actual efficacy of a potentially effective therapeutic compound isbeyond the scope of the invention, as it is appreciated by the inventorsthat the safety and therapeutic efficacy of a compound must ultimatelybe determined by clinical trials in humans.

However, those skilled in the art can practice a method of the inventionso as to increase the likelihood that a therapeutic compound willactually be effective in preventing or treating the disease in aclinical setting. For example, the efficacy of a therapeutic compoundcan be generalized by repeating the method using cells from additionalgenetically related normal and diseased individuals. Such individuals,if desired, can be from the same family or genetically homogeneouspopulation initially used, or from different families or geneticallyrelated populations. Such individuals can exhibit, if desired, the sameor varying degrees of disease severity or risk as the individuals whosecells were initially used in the method.

Additionally, the efficacy of the compound can be further validated byrepeating the method using assays that detect alterations in one or moredifferent predetermined properties associated with the pathologicalcondition that the predetermined property initially assayed. Moreover,the method can be repeated using varying concentrations of a compound todetermine the minimally effective and least toxic concentration.Therefore, the method can be used to identify those compounds that aremost likely to be safe, effective and practical as therapeutics toprevent or treat a pathological condition.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Identification of Therapeutic Compounds in a GeneticallyDefined Setting

Many human diseases have their origin in malfunctioning signaltransduction pathways. A simple signal transduction pathway in a normalindividual is shown in FIG. 1A, with an interaction between a ligand anda receptor initiating a cascade of intracellular signals that results ina normal response in an assay of a predetermined property. A defect inany one of these molecules, such as a ligand, a receptor, or anintracellular signaling molecule, can cause a pathological condition,which can be detected by an alteration in the predetermined propertyindicative of the pathological condition. The genetic relatednessbetween a normal individual and a diseased individual greatly enhancesthe probability that all the molecular components in the signalingcascade are similar or identical, except the component that causes thepathological condition.

As shown in FIG. 1B, a pathological condition can result from adefective receptor, indicated by the altered shape of the receptor ascompared to the normal receptor in FIG. 1A. A compound that binds theabnormal receptor and allows it to interact with ligand, shown as atriangle, can restore normal signaling, as determined by a more normalresponse in an assay of a predetermined property. Other compounds in aplurality of candidate therapeutic compounds that do not restoreinteraction between the ligand and receptor will not affect the readoutof the predetermined property in the diseased cell.

EXAMPLE II Identification of Therapeutic Compounds Specific forDifferent Subtypes of a Pathological Condition

A phenotypically similar pathological condition in individuals ofgenetically heterogeneous backgrounds can be caused by differentunderlying molecular mechanisms. For example, as shown in FIG. 1C, aphenotypically similar pathological condition to the pathologicalcondition depicted in FIG. 1B can result from a defect in a differentcomponent of the signal transduction pathway, as indicated by thealtered (non-square) shape of the ligand. A compound effective in apatient with the type I subtype of the pathology, indicated by atriangle in FIG. 1B, would not be able to restore normal signaling to acell from a patient with the type II subtype of the pathology, and wouldnot be an effective therapeutic compound for treating the type IIsubtype of the pathology. However, a compound that allows the defectiveligand to interact with the receptor in a cell from a patient with thetype II subtype of the pathology would restore normal signaling, and isa potentially effective therapeutic compound.

The methods of the invention can readily be applied to identifyingpotential therapeutic compounds that are effective against each of thedifferent subtypes of the pathology, without prior knowledge of themolecular mechanisms that underly the different subtypes of thepathology. Thus, in the above example, cells from a genetically relatedpopulation consisting of normal individuals and diseased individualsexhibiting the type I form of the pathology would be used in a screen toidentify a therapeutic compound potentially effective against the type Iform of the pathological condition. Likewise, cells from a geneticallyrelated population consisting of normal individuals and diseasedindividuals exhibiting the type II form of the pathology would be usedin a screen to identify a therapeutic compound potentially effectiveagainst the type II form of the pathological condition.

The compounds so identified can be used as diagnostic reagents todetermine the subtype of a pathology in a patient exhibiting clinicalindications of the pathology. For example, the ability of a cell from apatient to give a normal response in an assay of a predeterminedproperty in the presence of a compound that has been determined, asdescribed above, to restore normal signaling in the type I subtype ofthe pathology, indicates that the patient has that subtype of thepathological condition. In contrast, the inability of a cell from apatient to give a normal response in an assay of a predeterminedproperty in the presence of the same compound indicates that thatpatient has a different subtype of the pathology. As described above,compounds that restore normal signaling in each of the differentsubtypes of the pathology can be determined, and these compounds can beused in diagnostic assays to identify the particular subtype of thepathology in each patient.

EXAMPLE III Development of Combination Therapies

Most cellular functions involve multiple signal transduction pathways.Thus, the response in an assay of a predetermined property oftenrequires the function of several convergent signaling pathways. Such asituation is shown in FIG. 2, where two ligands each bind their ownreceptor and initiate signaling cascades that converge to produce asignal in an assay of a predetermined property. In this example, theproduct of gene X, which carries out a process involved in one signalingtransduction pathway, is a known disease associated gene. The productsof genes Y and Z carry out processes involved in a second signaltransduction pathway, and can be considered modifying factors of theeffect of gene product X in an assay of a predetermined property.

In a genetically homogeneous population in which the diseasedindividuals have disease allele X, and normal individuals have wild-typeallele X, the activities of gene products Y and Z are likely to beconstant within the population, and the measurement of the predeterminedproperty thus reflects the activity of X in each individual in thatpopulation. In contrast, in an outbred population, the measurement ofthe predetermined property reflects a varying contribution of theactivities of X, Y and Z in each individual. Therefore, in a geneticallyhomogeneous population, a therapeutic compound that targets gene productX can be identified. Additional screens in the presence of this compoundcan then be performed to identify compounds that affect the activitiesof modifying factors Y or Z, as measured by a further alteration in thereadout in an assay of the predetermined property. Therefore, theinvention provides a method of developing combination therapies thattarget both disease associated genes and their modifying factors.

In a different genetically homogeneous population, having differentalleles or activities of modifying factors Y and Z, a similar screenperformed in the presence of a compound that targets gene product Xwould likely identify different compounds that affect the activities ofthe modifying factors Y or Z in that population. Therefore, theinvention provides a method of developing combination therapies that arespecific for a patient's particular X, Y, and Z allele or activitycombination.

EXAMPLE IV Pathway Mapping of Disease-Associated Genes

In searching for a genetic basis for disease, methods have beendeveloped to scan the genes of multiple individuals in a pedigree and toassociate a marker on a locus with the disease history of the members ofthe pedigree. Such association are then used to map thedisease-associated genes to a particular locus. The methods describedherein can be used in a similar manner to map disease-associated genesto a “locus” of a signal transduction pathway.

As described herein, assays are known in the art that can be used tomeasure a predetermined property indicative of an alteration in any stepof a signaling pathway, such as receptor/ligand interaction, secondmessenger signaling, phosphorylation events, gene expression and thelike. For example, as shown in FIG. 3, three different predeterminedproperties representing three different loci within of a signalingpathway can be assayed. In a genetically homogeneous population, normalindividuals and diseased individuals will have few differences in themolecules that carry out signaling steps, apart from the moleculeresponsible for the disease pathogenesis. Thus, by comparing severalpredetermined properties in normal and diseased cells from individualsin a genetically homogeneous population, the step in the signalingpathway that is altered in diseased individuals can be determined, andthe disease-associated gene mapped to that “locus” of the signalingpathway. The disease-associated gene or gene product is likely to be anappropriate target in a high-throughput drug screening assay to identifycompounds that can be used to treat the disease.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1. A method of identifying a therapeutic compound potentially effectiveagainst a predetermined pathological condition, comprising: (a)contacting ex vivo a cell indicative of said pathological condition froma diseased individual with a plurality of candidate therapeuticcompounds under suitable assay conditions; (b) contacting ex vivo acontrol cell from a normal individual genetically related to saiddiseased individual with said plurality under said assay conditions,said genetically related normal individual having a common ancestorwithin 7 to 10 generations with said diseased individual, and (c)identifying a compound from said plurality that preferentially altersone or more predetermined properties of said cell from said diseasedindividual, said compound being characterized as a therapeutic compoundpotentially effective against said pathological condition.
 2. The methodof claim 1, wherein step (a) comprises contacting a cell from each oftwo or more genetically related diseased individuals.
 3. The method ofclaim 2, wherein said two or more genetically related diseasedindividuals exhibit a range of disease severity or risk.
 4. The methodof claim 1, wherein step (b) comprises contacting a control cell fromeach of two or more genetically related normal individuals having acommon ancestor within 7 to 10 generations with said diseasedindividual.
 5. The method of claim 1, wherein step (a) comprisescontacting a cell from each of two or more genetically related diseasedindividuals and step (b) comprises contacting a control cell from eachof two or more genetically related normal individuals having a commonancestor within 7 to 10 generations with said diseased individual. 6.The method of claim 1, wherein said normal individual having a commonancestor within 7 to 10 generations with said diseased individual andsaid diseased individual are members of a genetically homogeneouspopulation.
 7. The method of claim 6, wherein said geneticallyhomogeneous population is an Icelandic population.
 8. The method ofclaim 1, wherein said plurality of candidate therapeutic compoundscomprises greater than 10⁵ compounds.
 9. The method of claim 1, whereinsaid plurality of candidate therapeutic compounds are sequentiallycontacted with said cell from said diseased individual or said controlcell from said normal individual having a common ancestor within 7 to 10generations with said diseased individual.
 10. The method of claim 1,wherein said plurality of candidate therapeutic compounds aresequentially contacted with said cell from said diseased individual andsaid control cell from said normal individual having a common ancestorwithin 7 to 10 generations with said diseased individual.
 11. The methodof claim 1, wherein said plurality of candidate therapeutic compoundsare simultaneously contacted with said cell from said diseasedindividual or said control cell from said normal individual having acommon ancestor within 7 to 10 generations with said diseasedindividual.
 12. The method of claim 1, wherein said plurality ofcandidate therapeutic compounds are simultaneously contacted with saidcell from said diseased individual and said control cell from saidnormal individual having a common ancestor within 7 to 10 generationswith said diseased individual.
 13. The method of claim 1, wherein saidpathological condition is selected from the group consisting of diseasesof the cardiovascular system, nervous system, immune system, respiratorysystem, gastrointestinal system, endocrine system, and cancer.
 14. Themethod of claim 1, wherein said method is automated.
 15. The method ofclaim 1, wherein step (a) comprises contacting a cell from each of 10 ormore genetically related diseased individuals.
 16. The method of claim1, wherein step (a) comprises contacting a cell from each of 100 or moregenetically related diseased individuals.
 17. The method of claim 1,wherein step (b) comprises contacting a control cell from each of 10 ormore genetically related normal individuals.
 18. The method of claim 1,wherein step (b) comprises contacting a control cell from each of 100 ormore genetically related normal individuals.
 19. The method of claim 1,wherein said plurality of candidate therapeutic compounds comprisesgreater than 50 compounds.
 20. The method of claim 1, wherein saidpathological condition is cancer.
 21. The method of claim 1, whereinsaid predetermined property is selected from the group consisting ofproliferation, adhesion, differentiation, motility and apoptosis. 22.The method of claim 1, wherein said cell is obtained from a tissueselected from the group consisting of breast, prostate, colon, lung,brain and ovary.
 23. The method of claim 1, wherein said cell from saiddiseased individual is an unaffected cell.
 24. The method of claim 1,wherein said cell from said diseased individual and said cell from saidnormal individual are propagated in culture.
 25. The method of claim 1,wherein said cell from said diseased individual and said cell from saidnormal individual are transduced or transfected with a nucleic acidmolecule.
 26. The method of claim 1, further comprising: (d) repeatingsteps (a) and (b) with said therapeutic compound obtained from step (c),and determining the ability of said therapeutic compound topreferentially alter a second or more predetermined property of saidcell from said diseased individual.